1. Field of the Invention
The present invention relates to a method for detecting hybridization which utilizes hybridization between a sample DNA and a probe DNA to analyze whether a DNA sequence of interest is present in the sample DNA or not.
2. Description of the Prior Art
Heretofore, to identify and/or fractionate a molecule found in a living body, in particular, to detect a DNA of interest or to detect presence or absence of a gene DNA, many methods have been employed in which a nucleic acid or protein having a known sequence is hybridized as a probe. In many cases, however, samples used in biochemical experiments are very small in quantity. Accordingly, in analyses thereof, very high sensitivity of detection is required. To cope with this, a probe has been labeled with a radioisotope and hybridized with a sample, and then an X-ray film or the like has been exposed to radiation therefrom to detect the hybridization, thereby performing identification of the sample.
However, a radioisotope is hazardous, and thus handling thereof must be strictly regulated. Accordingly, in recent years, highly sensitive detection methods have been developed which use fluorescence or chemiluminescence instead of a radioisotope. In particular, fluorescent methods have been utilized frequently to detect a sample on a plane because an emission pattern on a plane is recognized as an image by fluorescent methods more easily as compared with chemiluminescence methods. In fluorescent methods, a sample labeled with a fluorescent material is irradiated with a laser beam to excite the fluorescent material, and intensity distribution of fluorescence emitted from the fluorescent material is measured to thereby identify the sample. An example of a device for recognizing such an emission pattern is described in Japanese Examined Patent Publication No.3481/1996.
Further, methods have been known which comprises labeling a sample and a probe with different fluorescent materials, and performing hybridization therebetween, followed by detection of the hybridization through multi-step excitation. FIGS. 2(a) to 2(f) are a schematic representation illustrating principle of those methods. As shown in FIG. 2(a), a sample DNA 11 is labeled with a fluorescent material 13, and as shown in FIG. 2(b), a probe DNA 12 is labeled with a fluorescent material 14. The fluorescent material 13 and the fluorescent material 14 are different from each other in excitation wavelength and in fluorescence wavelength. The sample DNA 11 and the probe DNA 12 which have been labeled with the fluorescent materials 13 and 14, respectively, are mixed in a hybridization solution 15. The hybridization solution comprises formaldehyde, SSC (standard saline citrate: NaCl, trisodium citrate), SDS (sodium dodecyl sulfate), EDTA (ethylenediaminetetraacetic acid), and distilled water. Proportions of the ingredients vary depending upon nature of a DNA used.
If the sample DNA 11 and the probe DNA 12 are those having complementary strands, they are hybridized into a double-stranded structure as shown in FIG. 2(c). On the other hand, if the sample DNA 11 and the probe DNA 12 are those having uncomplementary strands, they remain uncombined as shown in FIG. 2(d). To detect hybridization between the sample DNA 11 and the probe DNA 12, i.e., to detect whether these DNAs are combined or not, these are irradiated with excitation light 16 from a continuous light source which has a wavelength that allows the fluorescent material 13 to emit light but does not allow the fluorescent material 14 to emit light. If the sample DNA 11 and the probe DNA 12 are hybridized, the fluorescent material 14 is present in the vicinity of the fluorescent material 13, and consequently, excitation energy 17 of the fluorescent material 13 is transferred to the fluorescent material 14, and fluorescence 18 is emitted from the fluorescent material 14, as shown in FIG. 2(e). On the other hand, if the sample DNA 11 and the probe DNA 12 are not hybridized, the fluorescent material 13 with which the sample DNA 11 is labeled and the fluorescent material 14 with which the probe DNA 12 is labeled are distant from each other. Consequently, energy of the fluorescent material 13 cannot be transferred to the fluorescent material 14, and no fluorescence is emitted from the fluorescent material 14, as shown in FIG. 2(f). By observing the wavelength of the fluorescence emitted from the analyte in this manner, it is possible to detect the hybridization between the sample DNA 11 and the probe DNA 12.
Although a fluorescent probe is easy to handle, fluorescence emitted from a fluorescent labeling material is faint and thus its intensity is low. Further, the fluorescence has a wavelength which is not so different from that of excitation light. Accordingly, the excitation light cannot be cut off completely even if an excitation light cut-off filter is disposed in front of a detector. Therefore, highly sensitive detection has not been attained.
When such a probe for multi-step excitation is used, it is possible to increase difference between a wavelength of excitation light and that of fluorescence. This is advantageous in that detection is conducted with the fluorescence apart from the excitation light in wavelength. However, a sample DNA itself must be labeled with a fluorescent material. In a case where a sample DNA is subjected to an artificial treatment such as labeling with a fluorescent material, if an error is made in the step of the artificial treatment, the analysis no longer has reliability at all. If the sample is of vital importance, the error is irretrievable. Further, when the fluorescence is measured, the excitation light partially enters a detector as well together with the fluorescence. Accordingly, it is difficult to increase a S/N ratio.